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Fluorescent probe for detecting microcystic toxin-LR in water

A microcystin and fluorescent probe technology, applied in the fields of molecular biology and microbiology, can solve the problems of long cycle time, cumbersome preparation of MC-LR monoclonal antibody, and inability to detect on-site

Inactive Publication Date: 2013-05-22
CHANGCHUN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The first two methods are either due to high cost, long cycle, and high technical requirements; or due to the complexity of the experimental steps, expensive instruments, time-consuming pretreatment, and inability to detect on-site. Therefore, the main detection method at present is immunoassay, including enzyme immunoassay and protein phosphoinhibition assay, etc.
The core technology of immunoassay is the preparation of MC-LR antibody, but the preparation of MC-LR monoclonal antibody is cumbersome and expensive

Method used

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  • Fluorescent probe for detecting microcystic toxin-LR in water
  • Fluorescent probe for detecting microcystic toxin-LR in water
  • Fluorescent probe for detecting microcystic toxin-LR in water

Examples

Experimental program
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Effect test

Embodiment 1

[0094] Using MC-LR Standards to Establish Standard Detection Curves for Fluorescent Probes

[0095] 3. Screening of specific affinity ligands (screening includes non-specific elution and specific elution)

[0096] (1) The first round of non-specific elution

[0097] a. Use 0.1mol / L, pH 8.6 NaHCO with the prepared MC-LR 3 The solution was prepared as 100 μg / ml MC-LR solution. Add 1ml of this solution to a sterile polystyrene Petri dish (60×15mm) for coating, and carefully rotate repeatedly until the surface of the Petri dish is completely wet. After completion, put the culture dish into a plastic box covered with wet gauze and incubate at 4°C for more than 12 hours.

[0098] b. After incubation for 12 hours, pour off the coating solution in the petri dish, and vigorously pat on the filter paper that has been irradiated by ultraviolet light to remove the residual coating solution. After removing the residual liquid, add 2ml of blocking solution to the Petri dish, and block a...

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Abstract

The invention discloses a fluorescent probe for detecting microcystic toxin-LR in water, which belongs to the field of molecular biology and microbiology. According to the fluorescent probe, MC-LR is taken as a target, a dodecapeptide library randomly shown on the surface of a bacteriophage is used for selecting a recombination bacteriophage which is combined with the MC-LR in a specific manner, and the single-stranded DNA (Deoxyribonucleic Acid) of the recombination bacteriophage is extracted and recombined for sequencing and sequence alignment, thereby obtaining an MC-LR specific affinity ligand sequence: His-Phe-Phe-Lys-Trp-His-Thr-Arg-Thr-Asn-Asp-Gln, and subsequently, obtaining the specifically combined fluorescent probe FITC-Acp-His-Phe-Phe-Lys-Trp-His-Thr-Arg-Thr-Asn-Asp-Gln through solid-phase polypeptide synthesis and fluorescent marks. The fluorescent probe has the beneficial effects that firstly, a completely new tool is provided for the specific identification and content detection of the MC-LR; and secondly, the fluorescent ligand probe can be used for qualitatively identifying the MC-LR and quantitatively detecting the content of the MC-LR in a sample.

Description

technical field [0001] The invention belongs to the field of molecular biology and microbiology, and relates to screening a ligand sequence with specific affinity for microcystin-LR by using a random display peptide library on the surface of a phage, synthesizing the ligand by artificial solid-phase synthesis, and Fluorescently labeled it to prepare a fluorescent probe for detecting microcystin-LR. Background technique [0002] Microcystins (MCs) are a class of hepatotoxins released by harmful cyanobacteria blooms with strong cancer-promoting effects. Drinking water and aquatic products polluted by MCs pose a huge threat to human health. Among them, Microcystin-LR (Microcystin-LR, MC-LR) is the most toxic of MCs, and it is a biotoxin that poses a potential threat to public drinking water safety. [0003] Studies have shown that MC-LR can cause death in poultry, livestock and wild animals. And low doses of this toxin in drinking water can cause liver damage and promote the ...

Claims

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Application Information

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IPC IPC(8): C09K11/06C07K7/08C07K1/06C07K1/04G01N21/64
Inventor 张淑华于源华廖晓林王晴
Owner CHANGCHUN UNIV OF SCI & TECH
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